Herpesviruses are associated with many human ailments. Gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are involved in the genesis of human lymphoid and other cancers. Gammaherpesviruses undergo both lytic and latent forms of infection. The gammaherpesviral life cycle can be studied by using a murine gammaherpesvirus-68 (MHV-68) as a model system. Since viruses have limited coding capacity, many of the viral proteins are evolved as multifunctional with several domains. To obtain high-resolution functional definition of herpesviral multidomain proteins, a high-throughput genetic manipulation method, which can allow precise disruption of a particular functional domain without affecting neighboring domains, is required. The overall goal of this proposal is to establish a quantitative high-resolution functional genetic profile of a gammaherpesvirus genome. A functional genetic profiling platform has been developed by combining a Mu-transposon mediated 15-nucleotide random insertional mutagenesis method and a quantitative, high-throughput capillary genotyping method. The principle of this method has first been established by determining the cis-elements in the MHV-68 origin of lytic replication (oriLyt) that are required for oriLyt replicatory function by using an oriLyt mutant library containing 15-nt random insertions. Second, the gammaherpesvirus encoded transcription factor RTA is required for initiating the lytic gene expression program during lytic infection and reactivation from latency. The feasibility of the method in the context of the intact viral genome has been documented with a functional profile of the MHV-68 RTA promoter cis-regulatory elements and RTA protein microdomains which are essential for virus replication. Developing this method will establish a foundation for high resolution functional profiling of the entire MHV-68 genome. This proposal will develop a genome-wide and high- resolution functional profile for MHV-68 viral replication in cell culture, which will greatly expedite functional genomic studies of herpesviruses. The functional profile of MHV-68 replication in the lung in mice will be determined as well. By comparing in vivo to in vitro functional profiles we will identify MHV-68 sequences specifically relevant to viral replication in vivo. PUBLIC HEALTH RELEVANCE: Herpesviruses are associated with many human ailments. Gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are involved in the genesis of human lymphoid and other cancers. Since viruses have limited coding capacity, many of the viral proteins are evolved as multifunctional with several domains. To obtain a high-resolution functional definition of herpesviral multidomain proteins, a high-throughput genetic manipulation method, which can allow precise disruption of a particular functional domain without affecting neighboring domains will be developed. The information will facilitate the identification new therapeutic targets against herpesvirus replication and associated diseases.